Mass spectrometric diagnosis of sepsis without blood culture

ABSTRACT

The invention relates to methods and instruments for the rapid detection and rapid mass spectrometric identification of microbial infective agents in blood or other body fluids. The invention recognizes that blood is not a good environment for the cultivation of microbes and provides a method which (a) largely destroys or dissolves the human particles in body fluids, such as erythrocytes and leukocytes in blood, without impairing the ability of the microbes to reproduce, (b) separates the microbial pathogens from the fluid, (c) cultivates them in a nutrient broth which contains none of the antimicrobial components of the body fluids, (d) separates them from the nutrient broth, and (e) identifies the microbes by a mass spectrum of the microbial proteins. The dissolution of the human particles also releases the microbes nesting in macrophages. The cultivation in an optically clear nutrient broth with optimum composition not only accelerates the propagation of the microbes compared to all other cultivation methods, but also makes it possible to continuously measure their quantitative growth starting from a low microbe density. This firstly allows the mass spectrometric identification to be carried out at the earliest possible time, secondly provides a positive detection of microbes far ahead of their identification, which can be lifesaving for the patient; and thirdly makes it possible to start the determination of resistances early.

PRIORITY INFORMATION

This patent application claims priority from PCT patent applicationPCT/EP2011/063120 filed Jul. 29, 2011, which claims priority to Germanpatent application no. 10 2010 033 105.8 filed Aug. 2, 2010, which arehereby incorporated by reference.

FIELD OF INVENTION

The invention relates to methods and devices for the rapid detection andrapid mass spectrometric identification of infective microorganisms inblood (sepsis) or other body fluids.

PRIOR ART

An “identification” of microorganisms (termed microbes for short below)means their classification in the taxonomic hierarchy: domain(eukaryotes, prokaryotes and archaea), kingdom (plants, animals, fungi),division, class, order, family, genus, species and subspecies. Theidentification of a microbe sample means determining at least the genus,generally the species, and if possible also the subspecies, which isimportant when different subspecies have a different pathogenicity, forexample. In a broader sense, an identification can also mean acharacterization in terms of other, more individual characteristics ofthe microbes, such as the resistance of a microbe to antibiotics.

Many species of microbe, including bacteria in particular, but alsoprotozoa, micro-algae and undifferentiated fungal cells like yeast, canbe mass spectrometrically identified with a high degree of certainty bytransferring small amounts of microbes from a colony grown on a nutrientmedium in the usual way onto a mass spectrometric sample support plate,where they are disintegrated by a sample preparation. The mass spectrumobtained from this sample particularly shows the masses and abundancesof the different soluble proteins which are present in the microbes insufficient concentration. The identity of the microbes is determinedfrom this mass spectrum with the aid of a similarity analysis withreference spectra from a reference library.

The identification of the microbes is particularly important in relationto infectious diseases, especially sepsis. In a sepsis, microbes arereleased into body fluids continuously or intermittently from a (usuallyhidden) source, mostly into the blood circulation, but also into thespinal canal. In case of a sepsis, it is extremely important to be ableto detect and identify the types of pathogen very quickly in order thatappropriate medical treatment can be provided immediately. The situationis alarming: in Germany more than 60,000 people die every year due tosepsis; it is the third most common cause of death. In the USA around230,000 people die annually of sepsis according to estimates from theCenter of Disease Control (CDC). The number of cases of sepsis hasincreased continuously in recent years by around 1.5 percent per annum.The mortality rate is more than 40 percent. Rapid identificationincreases the chance of survival.

In the conventional mass spectrometric identification method in globaluse today, the microbes are first cultivated to form colonies. Thenutrient medium for the cultivation is usually in an agar in a Petridish, which enables pure “isolates” to be cultivated in separate microbecolonies in hours, days or weeks, depending on the vigor of themicrobes. If the colonies are superimposed or mixed, it is possible toobtain isolated colonies again in the usual way in a second cultivation.The microbes transferred by means of a small swab from a selected colonyonto the mass spectrometric sample support are then disintegrated bysprinkling with a strongly acidified solution of a conventional matrixsubstance (usually α-cyano-4-hydroxy cinnamic acid, HCCA, but also 2,5dihydroxy benzoic acid, DHB) for ionization by matrix-assisted laserdesorption (MALDI). The acid (usually formic acid or trifluoro-aceticacid) attacks the cell walls, and the organic solvent (usuallyacetonitrile) of the matrix solution can penetrate into the microbialcells and cause the weakened cell walls to burst. The sample is thendried by evaporating the solvent, whereby the dissolved matrix materialcrystallizes. The soluble proteins of the microbes, and some othersubstances of the cell as well to a very small extent, are incorporatedinto the matrix crystals in the process.

The matrix crystals with the incorporated analyte molecules arebombarded with focused UV-laser pulses in a mass spectrometer,generating ions of the analyte molecules in the vaporization plasmas,and these ions can then be measured in the mass spectrometer, separatedby their ion masses. Time-of-flight mass spectrometers are usually usedfor this purpose. The mass spectrum is the profile of these analyteions, which are predominantly protein ions. The ions with the mostuseful information for an identification have masses of between approx.3,000 and 15,000 atomic mass units. In this method the protein ions arepredominantly only singly charged (number of charges z=1), which makesit possible to simply refer to the mass in of the ions, instead ofalways using the term “mass-to-charge ratio” m/z, as is actuallynecessary in mass spectrometry.

The profile of the soluble protein ions, i.e. the mass spectrum, is verycharacteristic of the microbe species concerned because every species ofmicrobe produces its own mixture of genetically determined proteins,each protein having a characteristic mass. The abundance of the moreconcentrated soluble proteins which can be detected massspectrometrically are also genetically determined to a large extent andonly depend to a minor degree on the nutrient conditions or the maturityof the colony. The protein profiles are just as characteristic for amicrobe species as fingerprints are for an individual person. Today,many public and private institutes are acquiring reliable reference massspectra for reference libraries which may be used for diagnosticpurposes in medicine. All diagnostic methods, however, have to beapproved by official institutions in accordance with the correspondingnational laws. The mass spectrometric identification method withvalidated libraries on validated instruments has been approved in anumber of states.

This mass spectrometric method of identification has proven to beextremely successful. The certainty of a correct identification is fargreater than with the microbiological identification methods currentlyin use. It has been possible to demonstrate that, for many hundreds ofdifferent species of microbe, the identification certainty was fargreater than 95 percent. In cases of doubt, where there were deviationsfrom current microbiological identification methods, genetic sequencinghas confirmed that the mass spectrometric identification was correct inthe majority of cases. Since relationships between microbe species canalso be identified from similarities between the mass spectra, it waseven possible to correct classifications of microbe species in thetaxonomic hierarchy with the aid of simple mass spectrometricidentifications, finally confirmed by the far more complicated DNAsequencing.

To identify the microbes, mass spectra are measured from around 2,000atomic mass units up to high mass ranges of 20,000 atomic mass units,although the mass signals in the lower mass range up to around 3,000atomic mass units are less usable because they can originate fromexternally attached coat peptides and other substances whose presence israther random and variable, such as diet-dependant fatty acids. The bestidentifications can be obtained by evaluating only the mass signals inthe mass range from around 3,000 to 15,000 atomic mass units. Theultra-sensitive mass spectrometers now used for this purpose have only alow mass resolution, which means that the isotope groups whose masssignals each differ by one atomic mass unit can no longer be resolved inthis mass range. Only the envelopes of the isotope groups are measured.

This method of identifying microbes requires a pure culture of microbes,a so-called “isolate”, in order to obtain a mass spectrum on which nosignals of other microbes are superimposed. It has been found, however,that mass spectra of mixtures of two microbial species can also beevaluated, and that both species of microbe are identified (see thedocument DE 10 2009 007 266 A1, M. Kostrzewa et al., for example). Theidentification certainty suffers only slightly. If more than two microbespecies are involved in the mass spectrum, or if these two microbespecies are present in very different concentrations, the identificationprobability and identification certainty decrease greatly.

Despite the high risk to life, sepsis involves only small numbers ofmicrobes per volume of body fluid; in adults with blood sepsis theyusually amount to only 0.5 to 10 microbes capable of reproduction permilliliter of blood. In infants, the densities can be significantlyhigher, since their immune resistance is not yet fully developed. Inadults, the microbes in the blood are combated by various mechanisms: bymacrophages after they have been identified by antibodies, for example,but also by endogenous antibiotics, such as the defensins. Sepsis occurswhen the defense mechanisms in the blood do not succeed in destroyingthese microbes far faster than they are supplied from the foci ofinfection; secondary foci can then form very rapidly, on the heartvalves, for example, in which case an immediate operation is usuallynecessary. In some clear body fluids such as cerebrospinal fluid, thenumbers of microbes per volume can be much higher than in the blood, soa direct separation without any prior propagation has a very good chanceof leading to a mass spectrometric identification.

With pathogens in body fluids the time until their identification iscritical if medical treatment is to be successful. The document WPO2009/065580 A1 (U. Weller 2007) describes a method to directly separateinfective agents from body fluids by centrifuging and mass spectrometricidentification. Microbes can be transferred from the precipitate(pellets) directly onto the mass spectrometric sample support plate, forexample. However, this analysis of the directly separated microbes isonly possible when high concentrations of microbes are present in thebody fluid, as is the case in the urine of patients with kidney diseaseor inflammation in the genitals, for example. If human cells are presentin the body fluid, they must be destroyed, for example by osmosis,before the microbes can be separated; other methods for destruction havebeen known for a long time, however.

For a sepsis in blood, a method known as “lysis centrifugation” has beenknown for over thirty years; this method almost completely dissolvesblood particles with the aid of a saponin (a weak surfactant)immediately after sampling, separates the microbes by cautiouscentrifugation and cultivates them on suitable culture media in Petridishes without the disturbing effects of the blood. Suitably preparedtubes (“Isolator™”) with saponin and other additives and correspondingtools have been produced and sold commercially for decades by severalcompanies, first by DuPont (Wilmington, USA), then by WampoleLaboratories, Cranbury, N.J., USA, and now also by Oxoid Limited,Basingstoke, Hampshire, England. Isolator™ is a trademark ofCarter-Wallace, Inc., New York, N.Y. 10105 USA.

The patent application US 20100120085 (J. Hyman et al.) claims a massspectrometric characterization and identification of microbes in asample by mass spectrometric scanning, where non-microbial cells aredestroyed by the known method of dissolution by surfactants; themicrobes are then separated by centrifugation and identified by massspectrometry. This document also belongs to the closest Prior Art.

The method of direct mass spectrometric analysis of the microbesseparated out of body fluids is only successful if the microbes arepresent in high concentrations well above 10⁴ microbes per milliliter.In practice this is only the case in urine, in strongly inflamed tissue,in suppurative foci or sometimes in cerebrospinal fluid or synovialfluid. In other body fluids such as blood or lymph the quantities ofmicrobes are generally only small, even with severe sepsis. Even inacute sepsis there are less than ten microbes in one milliliter ofblood. This small quantity is not sufficient for a direct massspectrometric identification, so a cultivation stage to multiply themicrobes must be used beforehand.

The so-called “blood culture” in special blood culture bottles, whichare sold commercially in huge numbers, has been used for decades to growmicrobes in blood. This involves adding suitable nutrients andanticoagulants to the blood, as well as inhibitors or adsorbents for anyantibiotics which were administered to the patient before the blood wassampled. The blood culture bottles from different manufacturers differfundamentally in the type of adsorbents for these antibiotics. Charcoalor open-pored plastic foam beads are used, for example. Both also impedethe growth of the microbes, however. Usual practice is to use two bloodculture bottles at the same time, one for aerobic and one for anaerobicmicrobes. Modern blood culture bottles are provided with signal systemswhich, if used in appropriately equipped incubators, automaticallysignal when sufficient microbes for a microbiological identificationhave grown. The signals are based on measurement of the increase in theCO₂ content or the pressure, but the density of the microbes in blood isalready very high when the signal is triggered. Mass spectrometricidentification is usually possible hours or days before the signal istriggered, so at least a much more sensitive trigger mechanism isrequired for fast mass spectrometric identification.

Once a sufficiently large number of microbes has grown in the bloodculture, the microbes must be separated out by centrifugation orfiltration for a mass spectrometric identification. All traces of humanproteins and proteins from the nutrient medium must be removed in ordernot to interfere with the mass spectrometric identification.

When sufficient microbes have been separated, for example bycentrifugation after all human cells have been dissolved, the microbescan best be broken down by adding a few microliters of a strong acid(formic acid or trifluoroacetic acid) and around the same quantity ofacetonitrile; the solution with the released proteins can then betransferred onto the sample support plate. This dissociation of themicrobes is even possible if no visible precipitate has been produced bythe centrifugation. The visibility limit for a precipitate is around 10⁶microbes; the detection limit, in contrast, is currently around 10⁴microbes, but can probably be improved in the future. 10⁴ microbesusually contain more than 100 femtograms of soluble proteins, but themass spectrometric detection limit for an optimally developed method isfar below this.

The identification of the microbes in body fluids by mass spectrometryis successful because in the vast majority of cases (far more than 70percent) acute microbial infections are caused only by one singlespecies of microbe. Only a small percentage of cases—around 15percent—involve two microbe species such that both can be detected inthe mass spectra. This species purity of the pathogens of acuteinfections is in stark contrast to how microbes otherwise occur in or onthe human body; the approx. 10¹⁴ bacteria of the intestinal flora in thehuman intestine, for example, are distributed over at least 400bacterial species which live in equilibrium with each other. Blood andother body fluids are generally sterile, i.e. in the normal state theydo not contain any microbes whatsoever. Microbes in body fluids arenormally attacked, killed and removed immediately by several defensemechanisms operating simultaneously.

SUMMARY OF THE INVENTION

The blood culture described above is by no means an ideal nutrientmedium for microbes; a number of microbe species hardly grow at all inblood cultures, Blood is fundamentally hostile to microbes: apart frommicrobe-eating macrophages, it contains a number of antibioticsubstances, such as the defensins and antibacterial enzymes. Inaddition, the blood of patients with sepsis usually also containsbroad-spectrum antibiotics, which have been administered as the initialmedical treatment before blood is sampled for a diagnosis.

The invention recognizes this situation and provides a method which (a)largely destroys and dissolves the human particles in the body fluids,such as erythrocytes and leukocytes in blood, immediately after samplingif possible, while the microbes must still be kept able to reproduce,then (b) separates the microbial pathogens from the fluid bycentrifugation or filtration, for example, (c) cultivates them in anutrient broth which does not contain any of the antimicrobialcomponents of the body fluids, (d) separates them again from thenutrient broth, and (e) identifies them by analyzing the similaritybetween a mass spectrum of their protein profiles and reference spectra.

This fundamental method for the mass spectrometric identification of themicrobes can be accompanied by methods which monitor the quantitativegrowth of the microbes during the cultivation in Step (c), particularlyoptical methods. These methods offer an early detection for the presenceof microbes, but also a rough classification of the microbes. Opticalmonitoring is made possible only by the use of an optically clearnutrient broth, sometimes with a special formulation which assists themeasurement procedure. For example, it is possible to performspectrometric measurements such as light scatter analysis, extinctionmeasurements, fluorescence measurements in different regions of theelectromagnetic spectrum. Furthermore, acoustic or electrometricmonitoring methods can be applied. The cultivation can preferably becarried out in a microculture vessel with only very little nutrientbroth in order to increase the microbe density and thus the sensitivityof the accompanying monitoring method. Early intermediate results ofthese growth monitoring can be life-saving for the patient, but can alsoallow the method to be terminated if no growing microbes can bedetected. In particular the accompanying methods can determine theearliest possible point in time for carrying out the mass spectrometricidentification. The identification itself takes only a short time, andthus allows a targeted therapy to begin at an early stage.

An important aspect of the invention is also to release the bacterialspecies nesting in macrophages, such as mycobacteria or listerias.

After the microbes have been separated in Step (b) it is also possibleto interpose a detection method, such as nano-NMR or micro-Raman, inorder to detect whether any microbes at all are present. Today'sdevelopment of modern spectrometric or electrometric methods promisesfuture sensitivities which are so high that even the presence ofindividual microbes can be detected.

This method provides sufficient pathogens in a sufficiently pure formfor the mass spectrometric identification, and is faster than anypreviously known method. When the doctor treating the patient knows thespecies of microbe, he/she can immediately start with targeted treatmentbecause for most microbes it is known which antibiotics they willrespond to. It is often the case that the resistance situation, whichcan be different from region to region or from hospital to hospital, isknown for this microbe species. But it is also possible to use a portionof the cultivated microbes to determine their resistance to antibiotics.The early start of targeted and promising treatment of a patient who isin an intensive care unit with a sepsis, severe sepsis or septic shock,can not only save the life of the patient more often than is the casetoday, but early recovery also considerably reduces the costs of medicaltreatment because the patient has a shorter stay in intensive care.

BRIEF DESCRIPTION OF THE ILLUSTRATIONS

FIG. 1 depicts a cultivation vessel for methods according to thisinvention in the form of a centrifuge tube, whose cap contains a septumand a differential pressure measuring system. The pre-evacuatedcultivation vessel has a write-on panel with an additional barcode, aninterior coating which changes color to indicate an increase in CO₂, andcontains a solution which dissolves human particles.

FIG. 2 illustrates an example of a microculture vessel with inletcapillaries and, which are closed with the septa and. The microculturevessel with a volume of only around 100 microliters can be pre-evacuatedready for use so that microbes in a nutrient broth can easily beintroduced with a syringe through one of the septa. The growth ofmicrobes in the microculture volume can be monitored with externalmeasuring devices. It is possible to measure the extinction of the lightbeam from an external laser diode in a detector, for example, and toproduce a signal if sufficient microbes have grown. It is also possibleto monitor the scattered light of the light beam in a detector. Iffluorescent substances in proportion to the microbes can be excited bythe light beam, the fluorescent light might be monitored by detector.The extinction, scattered light or fluorescent light curves provideearly information on the presence of microbes. Other types ofspectrometry can be used in a similar way. The screw cap is used to openthe microculture vessel once the microbes have been separated bycentrifuging after the cultivation has finished. The microbes can bebroken down in the microculture vessel after removing the nutrientbroth, and the extraction liquid can be removed together with thedissolved microbe proteins.

FIG. 3 shows a very simple microculture vessel with septum, filled withabout 100-200 microliter of broth, within a monitoring arrangementconsisting of a lengthy light source and an extinction detector. Thecross section views in the bottom part of the figure illustrates thearrangement of the extinction detector, or detectors for scattered lightwhich is deflected with highest density in forward direction, ordetectors for fluorescent light which radiates in all directions withabout equal density. The tiny vessel is ideal for centrifugation aftercultivation, for washing, and for the disintegration of the pelletedmicrobes in an acidic matrix solution.

FIG. 4 presents a diagram of the basic procedural steps according tothis invention.

FIG. 5 presents a more detailed diagram of a preferred procedure withinserted steps.

FAVORABLE EMBODIMENTS

FIG. 1 depicts a cultivation vessel for methods according to thisinvention in the form of a centrifuge tube, whose cap contains a septumand a differential pressure measuring system. The pre-evacuatedcultivation vessel has a write-on panel with an additional barcode, aninterior coating which changes color to indicate an increase in CO₂, andcontains a solution which dissolves human particles.

FIG. 2 illustrates an example of a microculture vessel with inletcapillaries and, which are closed with the septa and. The microculturevessel with a volume of only around 100 microliters can be pre-evacuatedready for use so that microbes in a nutrient broth can easily beintroduced with a syringe through one of the septa. The growth ofmicrobes in the microculture volume can be monitored with externalmeasuring devices. It is possible to measure the extinction of the lightbeam from an external laser diode in a detector, for example, and toproduce a signal if sufficient microbes have grown. It is also possibleto monitor the scattered light of the light beam in a detector. Iffluorescent substances in proportion to the microbes can be excited bythe light beam, the fluorescent light might be monitored by detector.The extinction, scattered light or fluorescent light curves provideearly information on the presence of microbes. Other types ofspectrometry can be used in a similar way. The screw cap is used to openthe microculture vessel once the microbes have been separated bycentrifuging after the cultivation has finished. The microbes can bebroken down in the microculture vessel after removing the nutrientbroth, and the extraction liquid can be removed together with thedissolved microbe proteins.

FIG. 3 shows a very simple microculture vessel with septum, filled withabout 100-200 microliter of broth, within a monitoring arrangementconsisting of a lengthy light source and an extinction detector. Thecross section views in the bottom part of the figure illustrates thearrangement of the extinction detector, or detectors for scattered lightwhich is deflected with highest density in forward direction, ordetectors for fluorescent light which radiates in all directions withabout equal density. The tiny vessel is ideal for centrifugation aftercultivation, for washing, and for the disintegration of the pelletedmicrobes in an acidic matrix solution.

FIG. 4 presents a diagram of the basic procedural steps according tothis invention.

FIG. 5 presents a more detailed diagram of a preferred procedure withinserted steps.

FAVORABLE EMBODIMENTS

As presented in FIG. 4, the invention provides a method with thefollowing basic steps:

a) dissolving the human particles in the body fluid, preferably by weaksurfactants,b) separating the microbes from the fluid, by centrifugation orfiltration, for example,c) culturing the microbes in a nutrient broth which does not contain theantimicrobial components of the body fluids,d) separating the microbes from the nutrient broth, by centrifugation orfiltration, for example,e) identifying the microbes by comparing a mass spectrum of themicrobial proteins with reference spectra.

Preferred embodiments of the method, more detailed and specified, withinserted steps b1), b2), c1), c2) and d1) are shown in FIG. 5. Theadditional steps must not all be performed within the same embodiment.

The term “body fluids” is used in a broad sense to mean all internal orexcreted fluids of the body which can be infected with microbes. Apartfrom blood, they also particularly include lymph, synovial fluid,cerebrospinal fluid, urine, lachrymal fluid, homogenized tissue throughto fluids from suppurating abscesses or inflamed nasal mucus. In some ofthese fluids the microbe concentrations are so high that immediateidentification is successful, after the microbes have been cleanlyseparated, even without further cultivation. This invention does notconcern these fluids; microbes in these fluids can be directly separatedand identified with the aid of one of the methods in WPO 2009/065580 A1(U. Weller 2007), already cited above. In many of these body fluids,however, the microbe concentrations are so low that the microbes must behighly multiplied by cultivation before an identification can takeplace. This is where this invention comes in.

The method described in more detail below uses blood as the body fluid;it can, however, also be used for other body fluids, but the quantitiesof surfactants and other substances added must be adapted accordingly.The description here is limited to centrifugation as the means ofseparating the microbes from the fluid, but without wishing to excludefiltration or other separation methods, such as attachment to magneticbeads.

In principle, the blood corpuscles can be destroyed in Step (a) withdistilled water after prior centrifugation, which causes most of theblood corpuscles to burst due to osmosis. Repeating the centrifugationand addition of distilled water destroys almost all the bloodcorpuscles. The remaining cell membrane shreds, which are separated bycentrifugation together with the microbes, hardly disturb the subsequentcultivation in the nutrient broth.

The preferred method, however, uses weak surfactants for thedisintegration and solution of the blood particles in Step (a). It isparticularly important that the solution of the blood corpuscles isperformed by a precisely measured quantity of nontoxic, weak surfactantsin order that even sensitive microbes remain fully able to reproduce. Asolution with diluted, nontoxic saponin is favorable, for example. Thedelicate cell membrane of the blood corpuscles consists predominantly ofphospholipids, which join together in a non-covalent bond to form themembrane. Surfactants dissolve the non-covalent bonds of proteins andlipids. The phospholipids of the cell membranes are themselvesamphiphilic, i.e. they have the properties of surfactants, and can benano-colloidally dissolved by other surfactants by forming micelles,whereby the added surfactants bond to such a degree that they can hardlydamage the microbes farther.

An important aspect of the invention is also to release the bacterialspecies nesting in macrophages or other human cells of the blood, suchas mycobacteria or Listerias, by dissolving the blood corpuscles.

The dissolving of the blood corpuscles and the subsequent separation ofthe microbes from the blood should preferably be done immediately afterthe blood sample has been taken, because leaving sensitive microbes inblood for a prolonged period could make them unable to reproduce. Forsensitive microbes, the half life for the survival of viable microbes inblood is only a few hours, while robust microbes can even multiply inthe blood. But even in blood dissolved in saponin the half-life forsensitive microbes is not much longer; it is therefore recommended thatthe microbes be separated from the fluid by centrifugation or filtrationtwo hours after the blood corpuscles have been dissolved, at the latest.

In both cases—destruction with distilled water or dissolution with anexact amount of surfactants—different amounts of residues, such asincompletely dissolved cell walls and other non-soluble components ofthe blood corpuscles, may remain in the liquid, but do not significantlyinterfere with the remainder of the method. These components can beremoved later by washing the microbes separated in Step (d) in a specialwashing step (d1) with dissolution of all non-microbial proteins bystrong surfactants. In Step (a) it is important to destroy the bloodcorpuscles in such a way that all microbes embedded in cells arereleased. When the microbes are separated from the fluid in Step (b),all soluble human proteins are removed, including the soluble proteinsfrom blood corpuscles, together with all endogenous antibodies which areharmful for the reproduction of the microbes, such as defensins andantimicrobial enzymes. Defensins form up to 30 percent of the granularsubstance of neutrophilic granulocytes, which make up around 60 percentof the leukocytes. If necessary, washing steps (b1) may be inserted.

It is also particularly important to remove all broad-spectrumantibiotics already administered as part of the treatment of sepsispatients, which is done here automatically in Steps (b) and (b1). Thecommercially available blood culture bottles contain different types ofparticles to render these antibiotics harmless, ranging from charcoalfragments through to hard, open-pored plastic foam beads. Themanufacturers wage wars of words about these additions, but they allharm the unimpeded growth of the microbes in the blood cultures, partlybecause they firmly adsorb microbes, and partly because they literallygrind down the microbes when the blood culture bottles are continuouslymoved to and fro in the incubation chamber.

The previously mentioned intermediate washing step (d1) to clean themicrobes deposited after sufficient cultivation in Step (c) serves toremove all traces of residual foreign proteins which do not belong tothe microbes, i.e. all human proteins, and also all proteins whichoriginate from the nutrient broth and could be attached to the microbes.Soluble proteins could even have been produced by the living microbesfrom the undissolved cell walls of the blood corpuscles. If signals fromforeign proteins are superimposed on the signals of the microbe proteinsin the mass spectrum, an identification is made much more difficult, ifnot impossible. This cleaning in Step (d1) can be done with strongsurfactants such as SDS (sodium dodecyl sulfate) because for the massspectrometric identification, the microbes do not have to retain theirability to reproduce.

Finally, the microbes must be broken down and the soluble proteinsreleased in order to acquire the mass spectrum of the microbe proteinsfor the mass spectrometric identification in Step (e). As has alreadybeen described in the introduction, this disintegration of the microbescan preferably be done while they are still in the centrifuge tube witha few microliters of an aggressive organic acid such as formic acid ortrifluoroacetic acid and a few microliters of acetonitrile; but themicrobes can also be broken down, as is usually the case today, afterspreading some microbes onto a mass spectrometric sample support plate.Conventional methods are used to acquire the mass spectrum of themicrobe proteins and to identify them by similarity analyses withreference spectra in Step (e).

If centrifugation is used, the whole method from Step (a) to Step (d)and through to disintegration of the microbes to obtain a solution ofthe microbial proteins for acquiring the mass spectra can be performedin the same centrifuge tube, for example. A 1.5 milliliter standardcentrifuge tube can be used here, for example. But in order to detectsepsis with only 0.5 viable microbes per milliliter with sufficientstatistical certainty it is more advantageous to use special cultivationvessels with around 15 milliliter volume, in which eight to tenmilliliters of blood can be used. Their volume is similar to that of theblood culture bottles used up to now, but they should allowcentrifugation, which is not possible with blood culture bottles. The1.5 milliliter standard centrifuge tubes can be used in the analysis ofthe blood of infants, for example, because only small quantities ofblood are available in this case, and usually higher concentrations ofmicrobes are present in their blood in the event of sepsis. The methodis described here for the larger centrifuge vessels.

When a pre-prepared ready-to-use 15 ml centrifuge tube is used for thismethod, around eight milliliters of blood is added. The centrifuge tubeis preferably sealed by a septum and pre-evacuated; it is prefabricatedand contains two to four milliliters of a sterile solution whichcontains around 240 micrograms of nontoxic saponin, a small amount offoam inhibitor and at least one anticoagulant. Precise rules for thesterile filling of the blood are known from methods for the treatment ofblood cultures. The liquids in the centrifuge tube are mixed immediatelyby carefully swirling five times, and after approx. 30 secondscentrifuged for 10 minutes at 3000 g, whereby the microbes areprecipitated as a loose pellet. The supernatant is carefully removedwith a sterile syringe and disposed of a sterile capillary needle withmicrofilter can be inserted to allow aseptic air to subsequently flowin. Ten milliliters of a nutrient broth are now added to the microbes,again with a sterile syringe. The microbes are dispersed in the broth byagitation; the centrifuge tube is incubated for a specified time at aspecified temperature. Particularly advantageous is incubation withslight agitation in order to prevent the microbes from precipitating.

Commercially available blood culture bottles are usually provided withsignal devices which, in suitably equipped incubations chambers, canindicate when sufficient microbes have grown. These signal devices aresometimes based on wall coatings changing color when the CO₂ contentchanges, and sometimes on different types of measurement of the CO₂increase or simply on the indication of a pressure increase. When theCO₂ content is measured, two blood culture bottles must always be used:one for aerobic microbes and one for anaerobic microbes.

Although these signal devices have so far not been ideal for massspectrometric identifications because their sensitivity is too low, theycan also be applied in cultivation vessels for methods according to thisinvention. They even operate better here because the blood corpuscles donot release CO₂. FIG. 1 depicts such a cultivation vessel in the form ofa centrifuge tube 1, whose cap 2 contains a piercable septum 3 and amicro differential pressure measuring system 4. The cultivation vessel 1has a write-on panel 5 with an additional barcode 6; it is pre-evacuatedfor filling. As an alternative to the differential pressure measuringsystem (4), the tube can have an interior coating 7 which changes colorto indicate an increase in CO₂. The centrifuge tube contains a preciselymeasured quantity of a solution 8 which dissolves human particles andconsists of saponin, anticoagulants and foam inhibitors in sterilewater, for example. As above, sterile syringes and needles withmicrofilters are used for filling the tube and for exchanging fluids.Any contamination with microbes from the environment must be carefullyavoided until after the incubation. Two identical cultivation vesselswith different types of nutrient broth can be used for the cultivationof aerobic and anaerobic microbes.

After a sufficiently long incubation (Step c), the microbes are againseparated by centrifugation (Step d), three minutes at 10,000 g nowbeing sufficient if the cultivation vessels are designed for this. Thesupernatant is removed, and the microbes are taken up by shaking with a0.5 percent SDS solution in water in order to remove all remnants ofhuman or nutrient broth proteins (Step d1). After renewed centrifugationand removal of the supernatant, the microbes are washed with distilledwater and centrifuged again. It is important that all traces of SDS areremoved because SDS (and other strong surfactants) greatly impedes theionization of the proteins in the ion source of the mass spectrometer.

The disintegration of the microbes to release the soluble proteins, theacquisition of mass spectra and the identification of the microbes bytheir mass spectra (Step e) use conventional methods and are notdescribed in more detail here.

Instead of separating the microbes by centrifugation in Steps (b) and(d), they can also be separated by microfiltration and washed.Centrifuges are also usually used for the microfiltration. Since theaddition of surfactants almost completely dissolves the cell membranesof the blood particles and their inner structures, microfiltration alsoproduces a pure isolate of the microbes.

Experience shows that only around 15 percent of the blood cultures showa positive result. Since it is extremely advantageous for the survivalof a patient with suspected sepsis that the existence of sepsis isdetected as early as possible, a spectrometric detection method forprecipitated microbes, such as nano-NMR or micro Raman, can be insertedafter the first separation of the microbes in Step (b), for example.Ultra-sensitive microspectrometric and electrometric methods arecurrently being developed which aim to detect the presence of singlemicrobes in the centrifuge pellet. Early knowledge of the definiteabsence of microbes not only saves the cost of the further method, it isalso important for the attending physician, who can adjust his/hertreatment early on this basis. These detections have not yet succeeded,but could become very important in the future.

More promising, however, are embodiments of the method which monitor themultiplicative growth of the microbes in Step (c1), while they are beingcultivated in Step (c), with very high sensitivity by measuring themicrobial density. In Step (e1), the presence of microbes which arecapable of reproduction can be determined at an early stage by detectingan increase in the microbe density, and more importantly, the monitoringin Step (c1) can also determine the earliest time at which a massspectrometric identification can be started. The cultivation can then bestopped (Step c2). This monitoring only becomes possible by theinvention: replacing the blood culture with cultivation in a clearnutrient broth with defined optical characteristics. The term “clear”must not mean complete transparency in the full spectral range; it issufficient, when certain types of spectrometry can be applied withoutperturbation. In such a clear nutrient broth with customizedcomposition, different types of spectrometry can be used in wide rangesof the electromagnetic spectrum, for example infrared spectrometry,Raman spectroscopy, scattered light and absorption spectrometry, andmany more. Fluorescence spectrometry has particularly good detectionsensitivity, for example. This can be triggered or assisted by addingsubstances to the nutrient broth whose metabolism in microbes leads tofluorescent breakdown products, or whose attachment to the cell walls ofthe microbes causes measurable fluorescence. There are fluorescentsubstances which change their fluorescent wavelength by attachment tocell walls. Since the microbes also often grow on the walls of thecultivation vessel, surface measurement methods can also be used, suchas plasmon resonance spectrometry (PRS) or surface-enhanced Ramanspectrometry (SERS). It is also possible to use different types ofacoustic and vibrational spectrometry. Furthermore, electricalmonitoring methods can be used: measurement of the dielectric constantin suitably shaped vessels, for example. Often the different measurementmethods can not only track the growth, but also communicate a roughclassification of the microbes. Even the speed of growth is anindication for an initial classification, a classification which ismedically very relevant.

When using these measurement methods with monitoring of the microbe'sgrowth, it is particularly advantageous if the microbes separated inStep (b) are grown in a nutrient broth whose volume is much smaller thanthat of the blood. In a good nutrient broth, the microbes undergolargely unimpeded exponential growth up to a density of 10⁷ to 10⁸microbes per milliliter, while only around 10⁴ microbes are required forthe mass spectrometric identification. The cultivation can thereforetake place in micro-quantities below one milliliter: just 100microliters of nutrient broth, for example, is sufficient to grow enoughmicrobes for a mass spectrometric identification. This makes it possibleto transfer the microbes, in a Step (b2), into a specially shapedmicrocultivation chamber with a volume of only about 100 to 200microliters after a short starting phase of around one to two hours ofcultivation in the original centrifuge vessel, in only 100 microlitersof nutrient broth, for example. In this small volume of nutrient broththe inoculation density, and thus also the subsequent microbe density,is around 100 times higher at every stage of the cultivation than in anvolume that corresponds to the quantity of the blood. Examples for suchmicro-cultivation chambers are shown in FIGS. 2 and 3. Thesemicro-cultivation chambers are particularly suited to the differenttypes of spectrometry because the microbe density is 100 times higher.

With a medium reproduction rate of only one generation of microbes perhour, a 12-hour cultivation generates the required 10⁴ microbes from asingle microbe. Aggressive types of microbes, as for instance E. coli,reproduce much faster.

The micro-cultivation chamber 12 shown in FIG. 2 can be very simplyscanned with a for measuring the extinction of light: this devicemonitors the attenuation caused by light scattering or absorption when alight beam 16 is sent through the chamber. Light source 15 andextinction measurement unit 17 does not need to be connected to themicrocultivation chamber, but can be installed in a chamber for theincubation and thus cyclically scan several microcultivation chambers.It is not only possible to measure the extinction; a similar measurementdevice 19 can measure scattered light 18 or fluorescent light 18 whichoriginates from the growing microbes when they are irradiated with alight beam 16. Light source 15 and extinction measurement unit 17 doesnot need to be connected to the micro-cultivation chamber, but can beinstalled in a chamber for the incubation and thus cyclically scanseveral micro-cultivation chambers. It is not only possible to measurethe extinction; a similar measurement device 19 can measure scatteredlight 18 or fluorescent light 18 which originates from the growingmicrobes when they are irradiated with a light beam 16.

In the even simpler micro-cultivation chamber 12 shown in FIG. 4, thegrowth of microbes can be very simply monitored by light from a lengthydiode system 34 crossing the tube and detected with a device 34 formeasuring the extinction. Detectors 35 and 36 can easily detectscattered light (highest density in forward direction) or fluorescentlight (equal density in all directions).

If the micro-cultivation chambers have a favorable shape, thesemonitoring devices can, above all, give an accurate indication of whenapprox. 10⁴ microbes for the mass spectrometric identification havegrown. This achieves a time saving of many hours or even days comparedto signal devices normally used in blood cultures at present, which isoften crucial for the survival of the patient. As already mentioned,such a measurement system can also provide a reliable early indicationof the presence of microbes, and even a rough classification, which isalso of crucial importance for the early treatment of the patient. Thispossibility of cultivating in microcultivation chambers is also onlymade possible by the fundamental principle of this invention—thecultivation of the microbes in a special nutrient broth.

A microcultivation chamber similar to the ones shown in FIGS. 2 and 3 issuitable for many of the above-mentioned types of spectrometry, althoughthe shape and material of the walls and windows possibly have to beadapted to the type of spectrometry. It can also be shaped in such a waythat it is directly suitable for separating the microbes from the liquidby centrifugation, as in FIG. 3, and for finally disintegrating themicrobes, for example. It is also possible to use microcultivationchambers on silicon chips, and electric circuits to measure the microbegrowth can be present on the chip (“lab-on-the-chip”).

When particularly sensitive spectrometries are used for this monitoring,such as the above-mentioned fluorescence spectrometry with the use ofspecial substances, the growth of the microbes can be detected veryearly. In this case only microbes capable of growth, i.e. reallydangerous ones, are analyzed; this is in contrast to analyses of thepellet after Step (b), where microbes which are dead or otherwise unableto reproduce could also be measured. It cannot be emphasized oftenenough that the early detection of a real sepsis is of crucialimportance for the patient's chances of recovery, or even for theirchances of survival.

The invention is based in particular on cultivating the microbes not inthe antimicrobial body fluids, but in a particularly favorable nutrientbroth. As explained, it is the special nutrient broth which makes itpossible to also perform accompanying measurements of the microbegrowth. First, however, the microbes must be cleanly separated from thebody fluid. This is done using a method which is similar to onedeveloped more than thirty years ago for the Isolator™ tubes for theclean separation of microbes from blood, and which has meanwhile beentried and tested millions of times over. It involves quickly dissolvingthe different types of blood corpuscles in a precisely measured quantityof a weak surfactant without destroying the microbes or rendering themunable to reproduce. A purified, nontoxic saponin has proven to be agood weak surfactant, but many other types of surfactant can be used.The delicate cell membrane of the blood corpuscles consistspredominantly of phospholipids, which join together in a non-covalentbond to form the membrane. Surfactants dissolve the non-covalent bondsof proteins and lipids, but also destroy the quaternary and tertiarystructure of the macromolecular proteins in the interior of the cells byionic attachments. The phospholipids of the cell membranes arethemselves amphiphilic, i.e. they have surfactant characteristics, andcan be dissolved by other surfactants by forming nano-colloidalmicelles. As a result, the cell membranes largely dissolve, but thisalso binds the surfactants added to the blood to a large extent, afterwhich they can no longer damage the microbes. The internal structures ofthe blood corpuscles are also destroyed by surfactants and dissolved toa large extent, including the membrane of the cell nucleus and the DNAof the leukocytes. All dissolved components are removed with thesupernatant after the centrifugation. The macrophages are alsodissolved, thus releasing any microbes which may be nesting inside.

The cell walls of bacteria, on the other hand, are very sturdy; theyconsist mainly of cross-linked polymerized mureins. In the case ofgram-positive bacteria there is an additional cross-linking withteichoic acids, which are also polymerized. These covalently bondednetworks withstand the dissolving effect of the surfactants at least forthe short time of several minutes. Surfactants can also penetrate intothe microbes and destroy the folding structures of the proteins, whichimpairs their ability to reproduce; it is therefore important to use aprecisely measured quantity of a weak surfactant, preferably a nontoxicsaponin, for the first dissolution of the human particles in Step (a),and to only let the surfactant act for a short time.

The half-life for the survival of sensitive microbes in a saponin-bloodsolution is only a few hours. The microbes must therefore be separatedfrom the liquid in as short a time as possible. It is recommended thatthe microbes are not kept in the liquid for more than two hours. Thecultivation vessels filled with blood must therefore be transportedquickly by courier or special conveyor systems to the laboratory, wherethe further method can be carried out. It is also possible to send theblood to the laboratory in normal blood containers with anticoagulants,although here as well, the shortest possible delay is of the essence.

For the mass spectrometric identification of the microbes grown byincubation, it is not important whether the microbes are dead or viable,as long as the proteins in the interior are not released or changed intheir primary structure. It is therefore possible to use a much strongersurfactant, SDS (sodium dodecyl sulfate) for example, to cleanse thesemicrobes of all residues of foreign proteins in Step (d1). The strongsurfactant must subsequently be thoroughly washed out again, however,because even in traces it will hinder the ionization of the proteins forthe acquisition of the mass spectra.

In a further embodiment of the method according to the invention, theblood in the centrifuge tubes can first be centrifuged. The supernatantclear blood plasma is then removed and the deep-red deposit is taken upwith a weak surfactant solution: a 1 percent saponin solution, forexample. The deep-red precipitate, which contains not only the microbesbut also, in particular, the 5 million erythrocytes, 7 thousandleukocytes and 50 thousand thrombocytes from each milliliter of blood,is mixed with the added surfactant solution in a shaker, a process whichdestroys the cell membranes of the blood corpuscles and releases thesoluble proteins. The deep-red solution is now centrifuged again, thesupernatant remaining deep red this time and the precipitate, ifvisible, appearing pure white. The nutrient broth can now be added tothe precipitate and incubated.

In a modification of this embodiment, the deep-red precipitate of theblood corpuscles can also simply be taken up by distilled water. This initself destroys most of the blood corpuscles. Repeating thecentrifugation and taking up the deposit with distilled water producesenough clean microbes for the cultivation in the nutrient broth.

Before the spectrum acquisition in Step (e), the microbes aredisintegrated and the soluble microbe proteins extracted. To this end,several microliters of a 70 percent formic acid is added to theprecipitated microbes. The acid aggressively attacks the peptidoglycans(mureins) of the cell walls of the microbes and destroys the cellstructure. The same quantity of acetonitrile is then added in order todissolve as many proteins as possible. The solution is again centrifugedin order to precipitate the solid components, such as remnants of themicrobes' cell walls.

A mass spectrum must now be acquired from the dissolved proteins of themicrobes in the supernatant. This can be done in a variety of ways withseveral known ionization methods in several types of mass spectrometers,but up to now special MALDI time-of-flight mass spectrometers have beenused exclusively

For the conventional acquisition of the mass spectrum in a MALDItime-of-flight mass spectrometer, around one and a half microliters ofthe supernatant is now applied to each measurement sample spot of aMALDI sample support, and dried. Then around one and a half microlitersof a solution of matrix substance is added, preferably HCCA dissolved in50% acetone in water with the addition of a little (around 3%)trifluoroethanoic acid (TFA). For this purpose, sample supports are usedwhich contain hydrophilic anchors two millimeters in diameter in ahydrophobic environment, for example. But it is also possible to usedisposable sample supports, on which etched circles prevent the solutionfrom spreading. The solution forms hemispheric droplets two millimetersin diameter. After drying all the measurement samples, the samplesupport is ready for the acquisition of the mass spectra.

This most simple preparation method for the measurement samples can bemodified in a wide variety of ways. One option is to use sample supportplates which already carry a thin layer of the matrix substance: HCCA,for example. The supernatant of formic acid and acetonitrile is thenpipetted directly onto this thin layer. The thin layer has the propertyof being able to immediately bond all proteins on the surface, so afteraround one minute the remaining liquid can be removed. This also removesimpurities. The subsequent optional addition of a droplet of acetone canembed the proteins into the small crystals of the thin layer byre-crystallization processes.

After the cultivation is finished, the process of preparing themeasurement sample for the acquisition of a mass spectrum in a MALDItime-of-flight mass spectrometer takes only around 10 to 15 minutes intotal. The sample support with the sample preparations is now introducedinto the ion source of a commercially available mass spectrometer in theusual way via a vacuum lock. The mass spectrometer is ready foroperation in around five minutes. In a MALDI mass spectrometer whose UVpulse laser operates at 200 hertz, it takes only a few seconds toacquire a sufficient number of individual spectra from a measurementsample in order to obtain a very usable sum spectrum. The acquisition ofthe mass spectra can therefore be completed in a few minutes.

Computer programs are commercially available for the subsequentidentification of the microbes by means of their mass spectra. The timerequired for identifying the microbes from good mass spectra depends onthe power of the computer, the size of the library of reference spectra,and the algorithm for the similarity analysis. With commerciallyavailable computers in mass spectrometers, identification of the massspectra from the samples, including the confirmation samples, takes onlya few minutes; the microbe species is therefore identified half an hourafter cultivation of the microbes finishes, at the latest.

In a different embodiment, if the precipitate is visible after a finalwashing step, a small quantity of the microbes thus isolated can betransferred onto the sample support plate in the usual way by means of aswab, and can be prepared there as usual. The mass spectra of thisconventional swab technique are largely similar to the mass spectra ofthe digestion technique using acid in the centrifuge tube. If there areany discernible differences, mass spectra of both types of samplepreparation can be entered in the library as reference spectra.

The invention primarily provides a method for definite identification ofmicrobial pathogens in blood that is significantly faster than currentmethods, which operate almost exclusively with cultivation of themicrobes in blood culture vessels and subsequent cultivation in Petridishes. The invention cultivates the microbes not in the antimicrobialblood, but in a nutrient broth favorable for microbe growth, from whichthey are separated, washed, and immediately identified massspectrometrically. The nutrient broth also allows the use of methods fora sensitive monitoring of the microbe growth so that cultivation can bestopped when the 10⁴ microbes required for mass spectrometricidentification have grown. The mass spectrometric method can be used toperform an identification without any prior knowledge of the microbes,and leads directly to an identification at the level of the microbespecies or subspecies. No other identification method is as fast andreliable.

The last Step (e) in the identification of the isolated microbes thusobtained follow conventional methods, which are otherwise usually basedon isolating one type of microbe by cultivating a colony. The isolationhere occurs automatically because, with acute infections, only one or atmost two species of microbe are to be found as pathogens. This meansthat sufficient pure microbe cultures (isolates) are obtained. Even whentwo species of microbe are present, the method still workssatisfactorily.

As has been emphasized several times, rapid identification of themicrobes of sepsis is extremely important. If the species frequentlyoccur, the resistance situation is usually known, so there is no urgencyto determine the resistance. For microbes occurring less frequently, butalso for some of the frequent microbe species which occur in greatlyvarying resistance situations, knowledge of their resistance todifferent antibiotics and also, most importantly, knowledge on thestrength of this resistance may be necessary. In this case, smallportions of the microbe precipitates isolated in Step (d) can also beused to determine the resistance of the microbes with the usualfunctional methods of attempted cultivation in the presence ofprogressive concentrations of antibiotics. If unusual microbes aresuspected at an early stage, they can be first cultivated in thenutrient broth before the nutrient broth is divided up among individualmicroculture bottles. In one of these bottles, the microbes arecultivated for the mass spectrometric identification, in others for thecharacterization of their resistance to different antibiotics.

The method of this invention with fast separation of the microbes fromthe endogenous cells can also be applied to abscesses or other foci ofinflammation, since they also contain endogenous cells. One example ofthis is a suppurative focus, i.e. an accumulation of some living, somepartially digested microbes in a mixture with certain types ofleukocytes which combat them. Here too, the endogenous cells can bedissolved with surfactant solutions. A further example is nasal mucusobtained as a swab of the nasal mucosa, for which the identification ofthe microbes (particularly MRSA) is of very great interest. Such samplescan also be obtained from other mucous membranes. Although in most casesdirect separation provides enough microbes for an identification withthese samples, it is also possible to carry out cultivation in asuitable nutrient broth if necessary.

In the methods described above, the mass spectra of the microbes wereacquired in time-of-flight mass spectrometers with ionization bymatrix-assisted laser desorption (MALDI). This is usual, but notobligatory. Disintegration liquids of microbes with soluble proteins canalso be ionized by electrospraying, for example. This type of ionizationgenerates strong superimposition of multiply charged ions in the massrange of about 600 to 1,600 atomic mass units, which necessarily requirea mass spectrometer with high resolution. More advantageous here is themethod of chemical ionization at atmospheric pressure (APCI), which verypredominantly provides singly charged ions of the analyte substances.Time-of-flight mass spectrometers with orthogonal ion injection(OTOF-MS) can be used here, as can ion cyclotron resonance massspectrometers (ICR-MS) or other high-resolution mass spectrometers.

The different types of cultivation vessel described above for the methodaccording to this invention can also be produced and sold commercially.They can also be put together in a commercially saleable pack ofconsumables (kit) with, for example: sterile disposable syringes foremptying; ready-to-use nutrient broth, supplied in syringes, forexample; puncture needles with microfilters for sterile ventilation;disposable sample supports and the matrix substance. With knowledge ofthe invention, the methods described here can be modified by thoseskilled in the art in a wide variety of ways. Some of thesemodifications have already been described above, but there are certainlyfurther methods which, on the fundamental basis of cultivation in anutrient broth, can provide not only an early mass spectrometricidentification of the microbes but also generate further information onthe microbes at an early stage.

Although the present invention has been illustrated and described withrespect to several preferred embodiments thereof, various changes,omissions and additions to the form and detail thereof, may be madetherein, without departing from the spirit and scope of the invention.

What is claimed is:
 1. A method for the identification of microbes inbody fluids comprising the Steps: a) Dissolving the human particles inthe body fluid; b) Separating the microbes from the fluid; c)Cultivating the microbes in a nutrient broth; d) Separating the microbesfrom the nutrient broth; and e) Identifying the microbes by similarityanalysis of a mass spectrum of the microbe proteins and referencespectra.
 2. The method of claim 1, wherein the body fluid is blood. 3.The method of claim 1, wherein the body fluid is lymph, synovial fluid,cerebrospinal fluid or homogenized tissue.
 4. The method of claim 1,wherein the human particles are dissolved with a surfactant in Step a).5. The method of claim 1, wherein the human particles are dissolved inan aqueous solution of saponin with added foam inhibitor.
 6. The methodof claim 1, wherein after Step b) it is investigated whether microbeshave been separated, and the Steps c) to e) are carried out only ifmicrobes have been detected.
 7. The Method of claim 1, wherein in Stepc) cultivation of the microbes takes place in less than one milliliterof nutrient broth.
 8. The method of claim 1, wherein the quantitativegrowth of the microbes is monitored during the cultivation of themicrobes in Step c).
 9. The method of claim 8, wherein the cultivationof the microbes in Step c) is carried out only until the monitoringindicates sufficient microbes for a mass spectrometric identification.10. The method of claim 8, wherein the monitoring of the quantitativegrowth of the microbes is carried out by measuring light extinction,scattered light or fluorescence.
 11. The method of claim 10, wherein forthe monitoring of the quantitative growth of the microbes byfluorescence, a substance is added to the nutrient broth whose breakdownby the microbes or attachment to the microbes causes a measurablefluorescence or a measurable change of the fluorescence wavelength. 12.The method of claim 1, wherein the microbes separated from the nutrientbroth in Step d) are cleansed of foreign proteins by an aqueous solutionof SDS (sodium dodecyl sulfate).
 13. The method of claim 1, wherein forthe acquisition of the mass spectrum in Step e) the microbe proteins areionized by matrix-assisted laser desorption (MALDI).
 14. The method ofclaim 1, wherein a portion of the microbes growing in the broth cultureis used for tests of their resistance to antibiotics.
 15. A cultivationvessel for carrying out the method of claim 1, suitable for separatingthe microbes by centrifugation.
 16. The cultivation vessel of claim 15,enabling external measuring devices to monitor the growth of themicrobes during cultivation.
 17. The cultivation vessel of claim 15,pre-evacuated for filling with the body fluid and containing a solutionof surfactants, foam inhibitor and anticoagulants for dissolving humanparticles in Step a).
 18. A pack of consumables, containing ready-to-usecultivation vessels according to claim 15 and ready-to-use nutrientbroth.